Ubiquitin (Ub) is a highly conserved eukaryotic protein that is involved in intracellular protein degradation, cell cycle regulation, stress response, the mechanism of receptor action and ribosome biogenesis. In all cases it appears to exert its function by being covalently attached to target proteins by an amide bond between the carboxyl terminal glycine of ub and an alpha- or epsilon-amino group on the attached protein. Ubiquitin carboxyl terminal hydrolases (UCH) are a class of cytoplasmic thiol proteases with specificity for cleavage of esters and amides of the carboxyl terminal glycine of ub and are required for the processing of all known ub gene products and metabolites. UCH isozymes are members of a newly recognized family of evolutionarily related proteins, and their expression is controlled in a tissue-specific manner. Two ubiquitin gene products must be proteolytically processed by UCH and are required for ribosomal function, and consequently cell growth. Ub must also be alternately removed from and attached to histones for cells to progress through the cell cycle. A defect in the regulation of UCH isozymes might contribute to a loss of growth regulation such as occurs in most cancers. Ub has been found on the cell surface and as a component of certain cell surface receptors. If the state of ubiquitination is important to receptor function, a defect in the expression of UCH could have an important effect of receptor affinity, receptor recycling, or signal transduction. UCH isozyme L1 is limited to neuronal and diffuse neuroendocrine tissues. Neurons have a vigorous stress response system, since accumulation of damaged proteins leading to cell death would have such a major import on the individual functions. The damage associated with stress due to stroke and reperfusion injury may be related to defects or insufficiencies in the ub-dependent proteolysis system. Over expression of an enzyme which removes ub from ubiquitinated proteins would be expected to interfere with the degradation of the damaged proteins. It is also important to study the expression of this isozyme in various neurological disease states. Several such neurodegenerative diseases, such as Alzheimers disease, Parkinsons disease, Progressive Supranuclear Palsy, and Picks' and Kufs' disease, are known to result in neurofibrillary tangles that are rich in ubiquitinated proteins. A defect or reduction in the expression of enzymes catalyzing the deconjugation of ub may be a characteristic of these disease states. The long term objectives of this proposal are to identify and characterize these related proteins, to determine the tissue distribution and regulation of the isozymes, to define the substrate specificity of these enzymes, and to elucidate the physiological role(s) for these enzymes. Specific aims include the purification, characterization and cloning of these enzymes, elucidation of the tissue distribution of these isozymes, and definition of the in vitro and in vivo substrate specificity.